Viral Replication Capacity Research Articles

Synonymous Codon Pair Recoding of the HIV-1 env Gene Affects Virus Replication Capacity.

Synonymous codon pair deoptimization is an efficient strategy for virus attenuation; however, the underlying mechanism remains controversial. Here, we optimized and deoptimized the codon pair bias (CPB) of the human immunodeficiency virus type 1 (HIV-1) envelope (env) gene to investigate the influence of env synonymous CPB recoding on virus replication capacity, as well as the potential mechanism. We found that env CPB deoptimization did not always generate attenuation, whereas CPB optimization attenuated virus replication in MT-4 cells. Furthermore, virus attenuation correlated with reduced Env protein production but not with decreased viral RNA synthesis. Remarkably, in our model, increasing the number of CpG dinucleotides in the 5′ end of env did not reduce the replication capacity of HIV-1. These results indicate that factors other than CPB or CpG content may have impacted the viral fitness of the synonymously recoded study variants. Our findings provide evidence that CPB recoding-associated attenuation can affect translation efficiency. Moreover, we demonstrated that an increased number of CpGs in the 5′ end of HIV-1 env is not always associated with reduced virus replication capacity.

Subtype-specific differences in Gag-protease replication capacity of HIV-1 isolates from East and West Africa

BackgroundThe HIV-1 epidemic in sub-Saharan Africa is heterogeneous with diverse unevenly distributed subtypes and regional differences in prevalence. Subtype-specific differences in disease progression rate and transmission efficiency have been reported, but the underlying biological mechanisms have not been fully characterized. Here, we tested the hypothesis that the subtypes prevalent in the East Africa, where adult prevalence rate is higher, have lower viral replication capacity (VRC) than their West African counterparts where adult prevalence rates are lower.ResultsGag-protease sequencing was performed on 213 and 160 antiretroviral-naïve chronically infected participants from West and East Africa respectively and bioinformatic tools were used to infer subtypes and recombination patterns. VRC of patient-derived gag-protease chimeric viruses from West (n = 178) and East (n = 114) Africa were determined using a green fluorescent protein reporter-based cell assay. Subtype and regional differences in VRC and amino acid variants impacting VRC were identified by statistical methods. CRF02_AG (65%, n = 139), other recombinants (14%, n = 30) and pure subtypes (21%, n = 44) were identified in West Africa. Subtypes A1 (64%, n = 103), D (22%, n = 35), or recombinants (14%, n = 22) were identified in East Africa. Viruses from West Africa had significantly higher VRC compared to those from East Africa (p < 0.0001), with subtype-specific differences found among strains within West and East Africa (p < 0.0001). Recombination patterns showed a preference for subtypes D, G or J rather than subtype A in the p6 region of gag, with evidence that subtype-specific differences in this region impact VRC. Furthermore, the Gag A83V polymorphism was associated with reduced VRC in CRF02_AG. HLA-A*23:01 (p = 0.0014) and HLA-C*07:01 (p = 0.002) were associated with lower VRC in subtype A infected individuals from East Africa.ConclusionsAlthough prevalent viruses from West Africa displayed higher VRC than those from East Africa consistent with the hypothesis that lower VRC is associated with higher population prevalence, the predominant CRF02_AG strain in West Africa displayed higher VRC than other prevalent strains suggesting that VRC alone does not explain population prevalence. The study identified viral and host genetic determinants of virus replication capacity for HIV-1 CRF02_AG and subtype A respectively, which may have relevance for vaccine strategies.

Differential impact of various substitutions at codon 715 in region II of HSV-1 and HCMV DNA polymerases

This study aimed at understanding the impact of different substitutions at codon 715 localized in the region II of the palm domain of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) DNA polymerases (pol). Here, we report a new theoretical mutation V715S that confers resistance of HSV-1 to foscarnet/acyclovir (5.6- and 9.2-fold increases EC50 values compared to wild type, respectively) and of HCMV to foscarnet/ganciclovir (2.8- and 2.9-fold increases in EC50 values compared to wild type, respectively). To further analyze the importance of this amino acid, we investigated the impact of the already known mutations V715M and V715G on the replicative capacities and drug susceptibilities of both viruses as well as on the activity and drug inhibition of the DNA pol. The V715G recombinant HSV-1 mutant was resistant to foscarnet and acyclovir (3.4- and 4.6-fold EC50 increase, respectively) whereas the V715M mutant was susceptible to foscarnet and resistant to acyclovir (3.4-fold EC50 increase). The V715G recombinant HCMV mutant did not grow and the V715M mutant was resistant to foscarnet (3.7-fold EC50 increase) and susceptible to ganciclovir. Finally, we showed by three-dimensional modeling that the differential impact of these mutations on the viral replicative capacity and drug resistance profile was related to different hydrophobic local environments for V715 in the DNA pol of the two viruses. Furthermore, we hypothesize that the DNA pol of HSV-1 is more tolerant to changes at this residue compared to that of HCMV because of a more hydrophobic environment stabilizing the region.

Mutation and Virulence of SARS-CoV-2 Strains Circulating in Anhui Province, China

Background: The genome of SARS-CoV-2 has shown considerable variation during its spreading. Monitoring variations in the virus genome to understand the evolution and spread of the virus is extremely important. Methods: Seven SARS-CoV-2 strains (BB127, BB183, HB030, MAS525, HF3028, FY1494, and SZ005) circulating in Anhui Province, China were isolated and sequenced for evolutionary analysis. Five strains were further cultured in vitro and were subjected to viral growth assay, TCID50 assay, and detection of spike protein expression. Next generation sequence (NGS) analysis were applied to investigate the mutation frequencies throughout the whole genome at different time gradients in vitro. Findings: Our observations revealed that in vitro cultured SARS-CoV-2 virus had much higher mutation frequency (up to ~20 times) than that in infected patients, and the mutation in nonstructural protein 14 (nsp14) might increase the genomic mutation frequency. Different strains had various amount of spike protein which may positively correlated with the virus replication capacity but may be influenced by other viral factors. Interpretation: Our study suggested that SARS-CoV-2 has the potential to diversify under favorable conditions. Monitoring viral mutations is not only helpful for better understanding of virus evolution and virulence change, but also the key to prevent virus transmission and disease progression. SARS-CoV-2 genomic variation analysis may also provide potential ideas for more efficient vaccine development and clinical treatment. Funding: This work is funded by Special Project for Emergency Scientific and Technological Research on New Coronavirus Infection (YG, No. YD9110002001), Emergency Research Project of Novel Coronavirus Infection of Anhui Province (Grant numbers 202004a07020002; 202004a07020004), Postdoctoral Research Foundation of China (2020M670084ZX) and the Fundamental Research Funds for the Central Universities (WK9110000166; WK9110000167).Declaration of Interests: We declare no competing interests.Ethics Approval Statement: The study was conformed to the principles of the Declaration ofHelsinki and approved by the Ethics Committee of the First Affiliated Hospital of USTC..

Abstract PO089: Comparison of Two oHSV Vectors for the Treatment of Glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common human brain cancer. Despite a well-established standard of care, the 5-year mortality rate of GBM patients is 95%, highlighting the need for innovative therapeutic interventions. A variety of oncolytic viruses, including those derived from herpes simplex virus (oHSV), have been designed for GBM therapy, but early-phase clinical trials have reported few complete responses without evidence of durable anti-tumor immunity. Potential reasons for the lack of efficacy are limited vector potency (i.e., virulence) and the presence of a highly immunosuppressive tumor microenvironment (TME) comprised of few activated lymphocytes, large numbers of immunosuppressive myeloid cells (macrophages, myeloid derived suppressor cells [MDSCs], microglia), and an agglomerate of immunosuppressive cytokines (IL-10, VEGF, MIF, etc.). Herein we explore these obstacles by comparing the anti-tumor activity of two different oHSV designs and by arming oHSV with an immunomodulatory payload designed to encourage the development of anti-tumor immunity. We first compared the activities of an HSV-1 KOS strain derivative designated KG4:T124 or colloquially, 2A5B, and an F strain derivative designated rQNestin34.5v.1 (a similar oHSV, rQNestin34.5v.2, is currently in a phase I clinical trial for GBM). We evaluated viral entry, replication capacity, and cytotoxicity using both murine and human glioma cell lines in vitro. Viral entry was relatively similar between both vectors, but rQNestin34.5v.1 replicated more effectively and generally induced greater viral mediated cytotoxicity. In syngeneic mice, rQNestin34.5v.1 reduced orthotopic GL261N4 tumor burden compared to 2A5B, but neither virus impeded disease progression in the more pernicious CT2A model. Evaluation of either the GL261N4 or CT2A TME revealed that both viruses induced a high influx of tumor associated macrophages, a transient polymorphonuclear (PMN) cell population, but did not alter the frequency of monocytic MDSCs. As a strategy to enhance oHSV efficacy, we sought to differentiate the highly immunosuppressive MDSC population into pro-inflammatory macrophages using oHSV armed with IL-12, a cytokine that has been shown to provide this activity. We show that oHSV:IL-12 induced a significant increase in tumor-associated macrophages and increased the survival of animals bearing either tumor type. Although we observed a low percentage of complete responses with the presence of anti-tumor CTLs demonstrated by tumor rechallenge experiments, IL-12 alone was not sufficient to significantly improve overall long-term animal survival, suggesting that additional arming genes should be considered in combination with IL-12. Citation Format: Joseph W. Jackson, Bonnie L. Hall, Lisa Bailey, Christophe Quéva, E. Antonio Chiocca, Justus B. Cohen, Joseph C. Glorioso. Comparison of Two oHSV Vectors for the Treatment of Glioblastoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO089.

Vulnerable targets in HIV-1 Pol for attenuation-based vaccine design

Identification of viral immune escape mutations that compromise HIV's ability to replicate may aid rational attenuation-based vaccine design. Previously we reported amino acids associated with altered viral replication capacity (RC) from a sequence-function analysis of 487 patient-derived RT-integrase sequences. In this study, site-directed mutagenesis experiments were performed to validate the effect of these mutations on RC. Viral reverse transcripts were measured by quantitative PCR and structural modelling was performed to gain further insight into the effect of reverse transcriptase (RT) mutations on reverse transcription. RT-integrase variants in or flanking cytotoxic T cell epitopes in the RT palm (158S), RT thumb (241I and 257V) and integrase catalytic core domain (124N) were confirmed to significantly reduce RC. RT mutants showed a delayed initiation of viral DNA synthesis. Structural models provide insight into how these attenuating RT mutations may affect amino acid interactions in the helix clamp, primer grip and catalytic site regions.

Contemporary circulating enterovirus D68 strains have acquired the capacity for viral entry and replication in human neuronal cells

Background: Enterovirus D68 (EV-D68) is a member of the Picornaviridae family of single-stranded positive-sense RNA viruses. EV-D68 infection has historically been associated with respiratory illnesses in humans. However, in the summers of 2014, 2016, and 2018 EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis (AFM) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. Methods and materials: To assess the neurotropic capacity of EV-D68, we explored the use of the neuroblastoma-derived neuronal cell line, SH-SY5Y, as a cell culture model to determine if differential infection is observed for different EV-D68 isolates. Viral replication and infectivity in SH-SY5Y was assessed using four different assays-infectious virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Results: In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to virus binding and entry were at least partly responsible for the differential infectivity phenotype, since transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains which could not directly infect SH-SY5Y. Conclusion: Taken together, these results suggest that EV-D68 has evolved to acquire novel neurotropic characteristics that may contribute to the development of neurological disease in humans. In addition to supporting virus replication and other functional studies, the cell culture model we’ve developed may help confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains.

Rescue of NanoLuc luciferase-expressing Senecavirus A with oncolytic activity

Senecavirus A (SVA), previously known as Seneca Valley virus, is classified into the genus Senecavirus in the family Picornaviridae. SVA is not pathogenic to normal human cells, but has potent oncolytic activity in some tumor cells with neuroendocrine feature, such as small cell lung cancer (SCLC) NCI-H446 cell line. In this study, we rescued and characterized a recombinant SVA that could efficiently express a novel luciferase, NanoLuc® luciferase (NLuc), which was smaller and “brighter” than others. This NLuc-tagged recombinant SVA (rSVA-NLuc) exhibited high capacity for viral replication, but genetic instability of NLuc during serial virus passages. The NLuc as a reporter facilitated oncolytic analysis of rSVA-NLuc in H446 cells. The rSVA-NLuc-infected H446 cells exhibited an oncolytic phenotype characterized by cell rounding, swelling, detachment and lysis at 48 h post infection. Kinetic curve showed that the NLuc was rapidly expressed in H446 cells during an exponential phase of viral growth. Because the NLuc offered several advantages over fluorescent proteins for assay scalability in vivo, the rSVA-NLuc would play a potential role in facilitating in vivo imaging studies of oncolytic virotherapy.

Progressive emergence of an S153F plus R263K combination of integrase mutations in the proviral DNA of one individual successfully treated with dolutegravir.

The development of HIV drug resistance against the integrase strand transfer inhibitor dolutegravir is rare. We report here the transient detection, by near full-genome ultradeep sequencing, of minority HIV-1 subtype B variants bearing the S153F and R263K integrase substitutions in the proviral DNA from blood cells of one patient who successfully initiated dolutegravir-based ART, over 24 weeks. Our objective was to study the effects of these substitutions. Strand transfer and DNA-binding activities of recombinant integrase proteins were measured in cell-free assays. Cell-based resistance, infectivity and replicative capacities were measured using molecular clones. Structural modelling was performed to understand experimental results. R263K emerged first, followed by the addition of S153F at Week 12. By Week 24, both mutations remained present, but at lower prevalence. We confirmed the coexistence of S153F and R263K on single viral genomes. Combining S153F or S153Y with R263K decreased integration and viral replicative capacity and conferred high levels of drug resistance against all integrase inhibitors. Alone, S153Y and S153F did little to infectivity or dolutegravir resistance. We identified altered DNA binding as a mechanism of resistance. The patient remained with undetectable viral loads at all timepoints. Drug-resistant minority variants have often been reported under suppressive ART. Our study adds to these observations by unravelling a progression towards higher levels of resistance through a novel pathway despite continuous undetectable viral loads. Poorly replicative HIV drug-resistant minority proviral variants did not compromise viral suppression in one individual treated with dolutegravir.

Quantitative Structure-Activity Relationship Study for HIV-1 LEDGF/p75 Inhibitors

HIV-1 Integrase (IN) is an important target for the development of the new anti-AIDS drugs. HIV-1 LEDGF/p75 inhibitors, which block the integrase and LEDGF/p75 interaction, have been validated for reduction in HIV-1 viral replicative capacity. In this work, computational Quantitative Structure-Activity Relationship (QSAR) models were developed for predicting the bioactivity of HIV-1 integrase LEDGF/p75 inhibitors. We collected 190 inhibitors and their bioactivities in this study and divided the inhibitors into nine scaffolds by the method of T-distributed Stochastic Neighbor Embedding (TSNE). These 190 inhibitors were split into a training set and a test set according to the result of a Kohonen's self-organizing map (SOM) or randomly. Multiple Linear Regression (MLR) models, support vector machine (SVM) models and two consensus models were built based on the training sets by 20 selected CORINA Symphony descriptors. All the models showed a good prediction of pIC50. The correlation coefficients of all the models were more than 0.7 on the test set. For the training set of consensus Model C1, which performed better than other models, the correlation coefficient(r) achieved 0.909 on the training set, and 0.804 on the test set. The selected molecular descriptors show that hydrogen bond acceptor, atom charges and electronegativities (especially π atom) were important in predicting the activity of HIV-1 integrase LEDGF/p75-IN inhibitors.

Infection with multiple HIV-1 founder variants is associated with lower viral replicative capacity, faster CD4+ T cell decline and increased immune activation during acute infection.

HIV-1 transmission is associated with a severe bottleneck in which a limited number of variants from a pool of genetically diverse quasispecies establishes infection. The IAVI protocol C cohort of discordant couples, female sex workers, other heterosexuals and men who have sex with men (MSM) present varying risks of HIV infection, diverse HIV-1 subtypes and represent a unique opportunity to characterize transmitted/founder viruses (TF) where disease outcome is known. To identify the TF, the HIV-1 repertoire of 38 MSM participants' samples was sequenced close to transmission (median 21 days post infection, IQR 18-41) and assessment of multivariant infection done. Patient derived gag genes were cloned into an NL4.3 provirus to generate chimeric viruses which were characterized for replicative capacity (RC). Finally, an evaluation of how the TF virus predicted disease progression and modified the immune response at both acute and chronic HIV-1 infection was done. There was higher prevalence of multivariant infection compared with previously described heterosexual cohorts. A link was identified between multivariant infection and replicative capacity conferred by gag, whereby TF gag tended to be of lower replicative capacity in multivariant infection (p = 0.02) suggesting an overall lowering of fitness requirements during infection with multiple variants. Notwithstanding, multivariant infection was associated with rapid CD4+ T cell decline and perturbances in the CD4+ T cell and B cell compartments compared to single variant infection, which were reversible upon control of viremia. Strategies aimed at identifying and mitigating multivariant infection could contribute toward improving HIV-1 prognosis and this may involve strategies that tighten the stringency of the transmission bottleneck such as treatment of STI. Furthermore, the sequences and chimeric viruses help with TF based experimental vaccine immunogen design and can be used in functional assays to probe effective immune responses against TF.

HIV-1 Lethality and Loss of Env Protein Expression Induced by Single Synonymous Substitutions in the Virus Genome Intronic-Splicing Silencer.

Synonymous genome recoding has been widely used to study different aspects of virus biology. Codon usage affects the temporal regulation of viral gene expression. In this study, we performed synonymous codon mutagenesis to investigate whether codon usage affected HIV-1 Env protein expression and virus viability. We replaced the codons AGG, GAG, CCU, ACU, CUC, and GGG of the HIV-1 env gene with the synonymous codons CGU, GAA, CCG, ACG, UUA, and GGA, respectively. We found that recoding the Env protein gp120 coding region (excluding the Rev response element [RRE]) did not significantly affect virus replication capacity, even though we introduced 15 new CpG dinucleotides. In contrast, changing a single codon (AGG to CGU) located in the gp41 coding region (HXB2 env position 2125 to 2127), which was included in the intronic splicing silencer (ISS), completely abolished virus replication and Env expression. Computational analyses of this mutant revealed a severe disruption in the ISS RNA secondary structure. A variant that restored ISS secondary RNA structure also reestablished Env production and virus viability. Interestingly, this codon variant prevented both virus replication and Env translation in a eukaryotic expression system. These findings suggested that disrupting mRNA splicing was not the only means of inhibiting translation. Our findings indicated that synonymous gp120 recoding was not always deleterious to HIV-1 replication. Importantly¸ we found that disrupting an external ISS loop strongly affected HIV-1 replication and Env translation.IMPORTANCE Synonymous substitutions can influence virus phenotype, replication capacity, and virulence. In this study, we explored how synonymous codon mutations impacted HIV-1 Env protein expression and virus replication capacity. We changed a single codon, AGG to CGU, which was located in the gp41 coding region (env nucleotide residues 2125 to 2127) and was included in the HIV-1 intronic splicing silencer. This change completely abolished virus replication and Env expression. We also found that changing codon usage in the gp120 region by including an increased number of CpG dinucleotides did not significantly affect Env expression or virus viability. Our findings showed that synonymous recoding was useful for altering viral phenotype and exploring virus biology.

Viral fitness of MHV-68 viruses harboring drug resistance mutations in the protein kinase or thymidine kinase

Murine γ-herpesvirus-68 (MHV-68), genetically and biologically related to human γ-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, can be easily propagated in vitro allowing drug resistance studies. Previously, we described specific changes in MHV-68 protein kinase (PK) or thymidine kinase (TK) associated with resistance to various purine or pyrimidine nucleoside analogues, respectively.To investigate how specific TK and PK mutations affect viral replication capacity, we performed dual infection competition assays in which wild-type and drug-resistant virus compete in absence or presence of antivirals in Vero cells. The composition of the mixed viral population was analyzed using next-generation sequencing and relative fitness of seven MHV-68 PK or TK mutants was calculated based on the frequency of viral variants at the time of infection and after 5-days growth.A MHV-68 mutant losing the PK function due to a 2-nucleotide deletion was less fit than the wild-type virus in absence of antivirals, consistent with the essential role of viral PKs during lytic replication, but overgrew the wild-type virus under pressure of purine nucleosides. TK mutant viruses, with frameshift or missense mutations, grew equal to wild-type virus in absence of antivirals, in accordance with the viral TK function only being essential in non-replicating or in TK-deficient cells, but were more fit when treated with pyrimidine nucleosides. Moreover, TK missense mutant viruses also increased fitness under pressure of antivirals other than pyrimidine nucleosides, indicating that MHV-68 TK mutations might influence viral fitness by acting on cellular and/or viral functions that are unrelated to nucleoside activation.

Hepatitis B virus mutation pattern rtA181S+T184I+M204I may contribute to multidrug resistance in clinical practice: Analysis of a large cohort of Chinese patients

The study aimed to characterize the prevalence and virological features of the rtA181S + T184I + M204I mutant in a large cohort of patients with chronic HBV infection. In total, 22,009 nucleoside/nucleotide analog-treated patients who underwent resistance testing at the Fifth Medical Center of Chinese PLA General Hospital between 2007 and 2016 were enrolled. Serum samples were collected for HBV reverse-transcriptase gene sequencing. Phenotypic analysis of the viral replication capacity and drug susceptibility was performed. The rtA181S mutation was detected in 0.82% (180/22,009) of samples. rtA181S-positive patients had significantly higher lamivudine (LAM), adefovir (ADV), and entecavir (ETV) exposure than rtA181S-negative patients. Of 180 rtA181S-positive patients, 42 had no coexistent resistance mutations, 34 had coexisting LAM-resistance mutation (LAMr), 17 had coexisting ADV-resistance mutation (ADVr), and 86 had coexisting ETV-resistance mutation (ETVr), and one had ADVr + ETVr. rtA181S + T184I + M204I occurred in 79.1% (68/86) of patients with rtA181S + ETVr and 37.8% (68/180) of all rtA181S-positive patients. Longitudinal analysis of the clinical course of resistant mutant evolution for four representative cases showed that rtA181S + T184I + M204I developed in all patients who had received LAM/telbivudine ± ADV and was receiving ETV or ADV + ETV. Compared with wild-type, the rtA181S + T184I + M204I mutant had 53.7% lower replication capacity and >1000-, 3.9-, and 383.3-fold greater LAM, ADV, and ETV resistance, respectively, but remained sensitive to tenofovir. Artificial elimination of rtA181S from the rtA181S + T184I + M204I mutant restored viral susceptibility to ADV but decreased viral replication capacity. Our study presented the first evidence that HBV rtA181S + T184I + M204I mutation had features of multidrug-resistance that contributed to resistance to both nucleoside and nucleotide analogs.

Impact of HLA-B*52:01-Driven Escape Mutations on Viral Replicative Capacity.

HLA-B*52:01 is strongly associated with protection against HIV disease progression. However, the mechanisms of HLA-B*52:01-mediated immune control have not been well studied. We here describe a cohort with a majority of HIV C-clade-infected individuals from Delhi, India, where HLA-B*52:01 is highly prevalent (phenotypic frequency, 22.5%). Consistent with studies of other cohorts, expression of HLA-B*52:01 was associated with high absolute CD4 counts and therefore a lack of HIV disease progression. We here examined the impact of HLA-B*52:01-associated viral polymorphisms within the immunodominant C clade Gag epitope RMTSPVSI (here, RI8; Gag residues 275 to 282) on viral replicative capacity (VRC) since HLA-mediated reduction in VRC is a central mechanism implicated in HLA-associated control of HIV. We observed in HLA-B*52:01-positive individuals a higher frequency of V280T, V280S, and V280A variants within RI8 (P = 0.0001). Each of these variants reduced viral replicative capacity in C clade viruses, particularly the V280A variant (P < 0.0001 in both the C clade consensus and in the Indian study cohort consensus p24 Gag backbone), which was also associated with significantly higher absolute CD4 counts in the donors (median, 941.5 cells/mm3; P = 0.004). A second HLA-B*52:01-associated mutation, K286R, flanking HLA-B*52:01-RI8, was also analyzed. Although selected in HLA-B*52:01-positive subjects often in combination with the V280X variants, this mutation did not act as a compensatory mutant but, indeed, further reduced VRC. These data are therefore consistent with previous work showing that HLA-B molecules that are associated with immune control of HIV principally target conserved epitopes within the capsid protein, escape from which results in a significant reduction in VRC.IMPORTANCE Few studies have addressed the mechanisms of immune control in HIV-infected subjects in India, where an estimated 2.7 million people are living with HIV. We focus here on a study cohort in Delhi on one of the most prevalent HLA-B alleles, HLA-B*52:01, present in 22.5% of infected individuals. HLA-B*52:01 has consistently been shown in other cohorts to be associated with protection against HIV disease progression, but studies have been limited by the low prevalence of this allele in North America and Europe. Among the C-clade-infected individuals, we show that HLA-B*52:01 is the most protective of all the HLA-B alleles expressed in the Indian cohort and is associated with the highest absolute CD4 counts. Further, we show that the mechanism by which HLA-B*52:01 mediates immune protection is, at least in part, related to the inability of HIV to evade the HLA-B*52:01-restricted p24 Gag-specific CD8+ T-cell response without incurring a significant loss to viral replicative capacity.

ER stress and autophagy induced by SARS-CoV-2: The targets for melatonin treatment

Coronavirus disease 19 (COVID-19) is a viral disease caused by the new coronavirus SARS-CoV-2. Like other coronaviral infections, SARS-CoV-2 causes oxidative and ER stress triggering cellular response pathways, mainly PERK and IRE1 branches of the UPR. This excessive oxidative stress and the increasing of unfolded and misfolded proteins induce autophagy. Once this process is triggered, the blockage of the fusion of autophagosomes and lysosomes induced by virus leads to an incomplete autophagy. Double-membraned vesicles, which create a membranous support for viral RNA replication complexes, are formed. Melatonin is a pleiotropic molecule, which reduces oxidative and ER stress, regulates immune system, and modulates autophagy pathway. Thus, melatonin reinforces UPR and unlocks autophagy blockage, allowing autophagosomes to bind to lysosomes, completing the process of autophagy and decreasing viral replication capacity. Based on these activities of melatonin the recommendation of melatonin for patients with COVID-19 should be seriously considered, especially in elderlies and patients with different comorbidities, which are the highest risk population for serious cases.

Association between the cytokine storm, immune cell dynamics, and viral replicative capacity in hyperacute HIV infection

IntroductionImmunological damage in acute HIV infection (AHI) may predispose to detrimental clinical sequela. However, studies on the earliest HIV-induced immunological changes are limited, particularly in sub-Saharan Africa. We assessed the plasma cytokines kinetics, and their associations with virological and immunological parameters, in a well-characterized AHI cohort where participants were diagnosed before peak viremia.MethodsBlood cytokine levels were measured using Luminex and ELISA assays pre-infection, during the hyperacute infection phase (before or at peak viremia, 1–11 days after the first detection of viremia), after peak viremia (24–32 days), and during the early chronic phase (77–263 days). Gag-protease-driven replicative capacities of the transmitted/founder viruses were determined using a green fluorescent reporter T cell assay. Complete blood counts were determined before and immediately following AHI detection before ART initiation.ResultsUntreated AHI was associated with a cytokine storm of 12 out of the 33 cytokines analyzed. Initiation of ART during Fiebig stages I–II abrogated the cytokine storm. In untreated AHI, virus replicative capacity correlated positively with IP-10 (rho = 0.84, P < 0.001) and IFN-alpha (rho = 0.59, P = 0.045) and inversely with nadir CD4+ T cell counts (rho = − 0.58, P = 0.048). Hyperacute HIV infection before the initiation of ART was associated with a transient increase in monocytes (P < 0.001), decreased lymphocytes (P = 0.011) and eosinophils (P = 0.003) at Fiebig stages I–II, and decreased eosinophils (P < 0.001) and basophils (P = 0.007) at Fiebig stages III–V. Levels of CXCL13 during the untreated hyperacute phase correlated inversely with blood eosinophils (rho = − 0.89, P < 0.001), basophils (rho = − 0.87, P = 0.001) and lymphocytes (rho = − 0.81, P = 0.005), suggesting their trafficking into tissues. In early treated individuals, time to viral load suppression correlated positively with plasma CXCL13 at the early chronic phase (rho = 0.83, P = 0.042).ConclusionWhile commencement of ART during Fiebig stages I–II of AHI abrogated the HIV-induced cytokine storm, significant depletions of eosinophils, basophils, and lymphocytes, as well as transient expansions of monocytes, were still observed in these individuals in the hyperacute phase before the initiation of ART, suggesting that even ART initiated during the onset of viremia does not abrogate all HIV-induced immune changes.

Construction of eGFP-Tagged Senecavirus A for Facilitating Virus Neutralization Test and Antiviral Assay.

Senecavirus A (SVA), also known as Seneca Valley virus, is an emerging virus that causes vesicular disease in pigs. This virus belongs to the genus Senecavirus in the family Picornaviridae. The SVA CH-LX-01-2016 was isolated from Guangdong Province of China in 2016. In this study, a recombinant SVA CH-LX-01-2016 was constructed using reverse genetics, and proven to be able to express efficiently an enhanced green fluorescent protein (eGFP) in vitro. This eGFP-tagged recombinant SVA (rSVA-eGFP) exhibited a high capacity for viral replication. Its fluorescence-tracked characteristics greatly facilitated both virus neutralization test (VNT) and antiviral assay. The rSVA-eGFP-based VNT was used to detect eight porcine serum samples, out of which four were determined to be neutralization titer-positive. Subsequently, two antiviral drugs, ribavirin and apigenin, were assayed for evaluating both effects against the rSVA-eGFP in vitro. The result showed that only the ribavirin exhibited an anti-SVA activity.

Mammalian pathogenicity and transmissibility of low pathogenic avian influenza H7N1 and H7N3 viruses isolated from North America in 2018

ABSTRACT Low pathogenic avian influenza (LPAI) H7 subtype viruses are infrequently, but persistently, associated with outbreaks in poultry in North America. These LPAI outbreaks provide opportunities for the virus to develop enhanced virulence and transmissibility in mammals and have previously resulted in both occasional acquisition of a highly pathogenic avian influenza (HPAI) phenotype in birds and sporadic cases of human infection. Two notable LPAI H7 subtype viruses caused outbreaks in 2018 in North America: LPAI H7N1 virus in chickens and turkeys, representing the first confirmed H7N1 infection in poultry farms in the United States, and LPAI H7N3 virus in turkeys, a virus subtype often associated with LPAI-to-HPAI phenotypes. Here, we investigated the replication capacity of representative viruses from these outbreaks in human respiratory tract cells and mammalian pathogenicity and transmissibility in the mouse and ferret models. We found that the LPAI H7 viruses replicated to high titre in human cells, reaching mean peak titres generally comparable to HPAI H7 viruses. Replication was efficient in both mammalian species, causing mild infection, with virus primarily limited to respiratory tract tissues. The H7 viruses demonstrated a capacity to transmit to naïve ferrets in a direct contact setting. These data support the need to perform routine risk assessments of LPAI H7 subtype viruses, even in the absence of confirmed human infection.

Characterization of envelope sequence of HIV virus in children infected with HIV in Vietnam.

Background:HIV is characterized by high levels of genetic variability, including increased numbers of heterogeneous sequences of the envelope region. Therefore, studying genetic variability of HIV in relation to viral replication might facilitate prognosis of disease progression.Methods:The study was designed as cross-sectional; data and samples of participants collected and analyzed env genes were obtained from 23 children enrolled by Vietnam National Children’s Hospital.Results:Substantial mutations in the C2 region were found in patients with high levels of viral replication while changes in the C3 region were mostly found in patients with low viral load. In the V1 region, we found profound amino acid modifications in patients with low HIV viral loads in contrast to the V2 sequence, where we identified single point mutations in patients with increased HIV viral load. The V3 region was relatively homogeneous, while profound deletions in the V4 region were detected in patients with increased viral replication.Conclusion:Our results suggest that genetic variations in different regions of the HIV envelope sequence, including both conserved C2 and C3 and variable V1/V2 and V4 regions, might be involved in increased viral infectivity and replication capacity. Such knowledge might help improve prediction of HIV progress and treatment in patients.